The objective of this project is to purify and study the enzymes involved in the interconversion of phylloquinone and phylloquinone-2,3-epoxide. Studies of these enzymes will be designed to determine their mechanisms and cellular requirements in order to determine if epoxidation of vitamin K is coupled to the carboxylation of glutamic acid residues in the precursor protein to form active prothrombin. Recent evidence has shown that the carboxylation reaction is the vitamin K-dependent step in the formation of prothrombin (Fernlund and Stenflo, 1975, J. Biol. Chem. 250, 6125). The possible role of vitamin K in carboxylation of protein other than those involved in blood coagulation will also be studied. The research goals of the coming year: (a) To determine the function of the cytosolic protein and NADH in epoxidation, prothrombin synthesis and carboxylation of glumatic acid in the precursor protein. (b) To determine the nature of the energy requirement in prothrombin synthesis and the mechanism of CO2 activation in the carboxylation reaction. (c) To determine the nature of the differences in reactivity when the quinone or hydroquinone of vitamin K is used as substrate for epoxidation and prothrombin synthesis. (d) To continue experiments designed to isolate the active components of phylloquinone exposidase and phylloquinone epoxide reductase from the microsomal fraction of rat liver. (e) To continue to study the effect of anticoagulants on the interconversion of phylloquinone and its 2,3-epoxide to determine on a molecular level the mechanism by which these compounds inhibit prothrombin synthesis. (f) To study the possibility that vitamin K and its epoxidation may be involved in other carboxylation reactions not associated with clotting protein synthesis.